Recent studies have revealed that MHC class Ib (MHC Ib) molecules contribute to the host immune response by presenting conserved microbial antigens to T cells. H2-M3 (M3), for example, presents N- formylated bacterial peptides to CD8+ cytotoxic T cells. However, the function of most MHC Ib molecules in immunity against intracellular bacterial infection remains largely unknown. Using mice that lack both MHC class Ia (MHC Ia) and M3 (Kb-/-Db-/-M3-/- mice), we found that non-M3 MHC Ib molecules are involved in antigen presentation to CD8+ T cells during Mycobacterium tuberculosis (Mtb) infection, although their exact role and the relative contribution of different MHC Ib molecules is yet to be determined. Unlike MHC Ia- restricted T cells, MHC Ib-restricted CD8+ T cells can exhibit a pre-activated phenotype in nave animals and respond rapidly to infection. Recently, we found that thymic selecting cell type plays an important role in determining the phenotype of M3-restricted T cells; hematopoietic cell-selected M3-restricted T cells acquire a more activated phenotype and possess more potent effector functions than those selected on thymic epithelial cells. However, it is still unclear how selecting cell type influences the phenotype of the resulting CD8+ T cells. One molecule that may play a role in regulating development of a pre-activated phenotype is SAP (SLAM-associated protein), which is known to be involved in the development of certain innate T cells. This proposal seeks to investigate the molecular and cellular mechanisms involved in regulating the development and function of MHC Ib-restricted CD8+ T cells, and to determine what roles these T cells play during Mtb infection. In Aim 1, we propose to determine whether SAP is required for hematopoietic cell- mediated selection of M3-restricted T cells and how SAP affects the effector functions of M3 and other MHC Ib-restricted CD8+ T cells. In Aim 2, we propose to characterize the repertoire and functionality of non-M3, MHC Ib-restricted CD8+ T cells in the immune response to aerogenic Mtb infection through the generation of T cell lines from infected Kb-/-Db-/-M3-/- mice. We will further assess the protective role of these CTL lines using an adoptive transfer approach. In Aim 3, we will focus on one MHC Ib molecule, Qa-1, as its human homolog HLA-E is known to present numerous Mtb peptides to CD8+ T cells, resulting in both cytotoxic and immunoregulatory activity. We propose to identify which HLA-E-binding Mtb peptides can also be presented by Qa-1 and examine the protective effect conferred by immunization with these peptides in Mtb infection. Further, we will characterize the immunoregulatory function of Qa-1 in anti-Mtb immunity, as well as the mechanisms responsible for this, using Qa-1-/- and Qa-1 mutant mice infected with Mtb. The results from our study will yield a better understanding of the developmental requirements, diversity and function of the MHC Ib-restricted immune response to Mtb, as well as potential new targets for Mtb vaccine development. PUBLIC HEALTH RELEVANCE: The MHC class Ib molecules comprise the majority of the MHC class I family but their role in anti- microbial immunity is relatively unknown. This study proposes to utilize novel animal models to examine the developmental requirements, regulation, and function of various MHC class Ib-restricted T cells in host defense against Mycobacterium tuberculosis, the causative agent of tuberculosis. Such studies may lead to identification of new targets for T-cell based vaccines against mycobacterial infection.